FirstGlance/Evaluating Protein Crosslinks: Difference between revisions
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Below are step-by-step instructions for evaluating putative [[protein crosslinks]] reported by FirstGlance in Jmol. Genuine crosslink bonds are illustrated, along with [[clashes]] that are irregularities in the model, not crosslink bonds. | Below are step-by-step instructions for evaluating putative [[protein crosslinks]] reported by FirstGlance in Jmol. Genuine crosslink bonds are illustrated, along with [[clashes]] that are irregularities in the model, not crosslink bonds. | ||
==Instructions for an Isopeptide Bond== | ==Instructions for an Isopeptide Bond== | ||
1. | 1. Go to [http://firstglance.jmol.org FirstGlance in Jmol] (right click link, open in new tab), and enter [[PDB code]] <b>2xi9</b>. | ||
<br> | <br> | ||
<table class="wikitable"><tr><td>[[Image:Fgij-xl1.png]]</td></tr></table> | <table class="wikitable"><tr><td>[[Image:Fgij-xl1.png]]</td></tr></table> | ||
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6. Finally, click on the link to show the ''Electron Density Map''. At the very good resolution of 1.9 Å, every atom in this pair of residues has clear electron density. There is no ambiguity about where these atoms belong in the model. Therefore, the electron density map supports the presence of this isopeptide bond. | 6. Finally, click on the link to show the ''Electron Density Map''. At the very good [[resolution]] of 1.9 Å, every atom in this pair of residues has clear electron density. There is no ambiguity about where these atoms belong in the model. Therefore, the electron density map supports the presence of this isopeptide bond. | ||
<br> | <br> | ||
<table class="wikitable"><tr><td>[[Image:Fgij-xl8.png]]</td><td>[[Image:Fgij-xl8.gif]]</td></tr></table> | <table class="wikitable"><tr><td>[[Image:Fgij-xl8.png]]</td><td>[[Image:Fgij-xl8.gif]]</td></tr></table> | ||
===Conservation of Crosslinking Residues=== | |||
If the PDB file is pre-processed by [[ConSurf/Index|ConSurf]], after displaying the crosslinked residues, their levels of evolutionary conservation will be shown by checking ''ConSurf Colors'' (in the middle-left of FirstGlance). | |||
'''For more complete instructions and illustrations, see [[Interpreting ConSurf Results]]'''. | |||
For example, below is a thioether covalent crosslink between Cys356 and Tyr379 in catalase [[1sy7]]. (The multiple sequence alignment included 150 sequences sampled from 22,855 unique similar sequences. The [[Interpreting_ConSurf_Results#Average_Pairwise_Distance|average pairwise distance]] was 0.96.) | |||
<table class="wikitable"><tr><td> | |||
[[Image:1sy7-fg-thioether.png]] | |||
</td><td width="350"> | |||
[[Image:1sy7-fg-consurf-thioether.png]] | |||
</td></tr><tr><td> | |||
Thioether crosslink in catalase [[1sy7]]. | |||
<br> | |||
<span style="font-size:160%;"> | |||
{{Template:ColorKey_Element_C}} | |||
{{Template:ColorKey_Element_N}} | |||
{{Template:ColorKey_Element_O}} | |||
{{Template:ColorKey_Element_S}} | |||
</span> | |||
</td><td> | |||
Crosslinking residues: | |||
<br> | |||
{{Template:ColorKey_ConSurf_NoYellow_NoGray}} | |||
<br> | |||
Heme: | |||
<span style="font-size:160%;"> | |||
{{Template:ColorKey_Element_C}} | |||
{{Template:ColorKey_Element_N}} | |||
{{Template:ColorKey_Element_O}} | |||
{{Template:ColorKey_Element_Fe}} | |||
</span> | |||
<br> | |||
Clicking the Cys sulfur atom (green arrow) produced the report at the bottom. | |||
</td></tr></table> | |||
Additional crosslink examples showing conservation: | |||
*[[FirstGlance/Visualizing_Conservation#Conservation_of_Protein_Crosslinks|An isopeptide crosslink in polyubiquitin]]. | |||
*[[Interpreting_ConSurf_Results#More_Sequences_Needed|An isopeptide in crosslinked lamprey fibrin]]. | |||
==Verified Crosslink Examples== | ==Verified Crosslink Examples== | ||
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==Clashes== | ==Clashes== | ||
FirstGlance suggests the presence of a "putative crosslink" based solely on the distances between relevant atoms (click the link '''Detailed Methods''' in FirstGlance). Sometimes, instead of a real covalent bond crosslink, the proximity of the relevant atoms is due to errors in the atomic model that produce [[clashes]]. | FirstGlance suggests the presence of a "putative crosslink" based solely on the distances between relevant atoms (click the link '''Detailed Methods''' in FirstGlance). Sometimes, instead of a real covalent bond crosslink, the proximity of the relevant atoms is due to [[alternate locations]] or small errors in the atomic model that produce [[clashes]]. | ||
For each putative crosslink suggested by FirstGlance, it is the responsibility of the user to evaluate whether it is a real covalent crosslink, or a clash. FirstGlance makes this responsibility clear (see the bottom paragraph in the snapshot under step #4 above). | For each putative crosslink suggested by FirstGlance, it is the responsibility of the user to evaluate whether it is a real covalent crosslink, or a clash. FirstGlance makes this responsibility clear (see the bottom paragraph in the snapshot under step #4 above). | ||
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===Bonds=== | ===Bonds=== | ||
It is important not to take the bonds displayed by FirstGlance/Jmol literally. [[Atomic coordinate files]] from the [[wwPDB]] do not specify most covalent bonds. Rather, Jmol draws covalent "bond" sticks between atoms wherever two atoms are sufficiently close. In the case of carbon, nitrogen and oxygen, this is wherever a pair of those atoms is separated by ≤1.81 Å<ref name="bondlengths">Actual covalent bond lengths are approximately C-C: 1.4 Å; C-N: 1.4 Å; C-O: 1.4 Å; C=O: 1.2 Å C-S: 1.7 Å([https://en.wikipedia.org/wiki/Covalent_radius Covalent radii in Wikipedia]).</ref>. In perfectly accurate models, this works correctly, but the errors in [[empirical models]] sometimes lead to bonds being drawn incorrectly. Thus, Jmol may show bond sticks where the reality is a clash, not a bond; and Jmol may fail to show bond sticks between atoms that are believed to be covalently bonded, but are too far apart in the model. | It is important not to take the bonds displayed by FirstGlance/Jmol literally. [[Atomic coordinate files]] from the [[wwPDB]] do not specify most covalent bonds. Rather, Jmol draws covalent "bond" sticks between atoms wherever two atoms are sufficiently close. In the case of carbon, nitrogen and oxygen, this is wherever a pair of those atoms is separated by ≤1.81 Å (for actual bond lengths, see footnote<ref name="bondlengths">Actual covalent bond lengths are approximately C-C: 1.4 Å; C-N: 1.4 Å; C-O: 1.4 Å; C=O: 1.2 Å; C-S: 1.7 Å ([https://en.wikipedia.org/wiki/Covalent_radius Covalent radii in Wikipedia]).</ref>). In perfectly accurate models, this works correctly, but the errors in [[empirical models]] sometimes lead to bonds being drawn incorrectly. Thus, Jmol may show bond sticks where the reality is a clash, not a bond; and Jmol may fail to show bond sticks between atoms that are believed to be covalently bonded, but are too far apart in the model. | ||
==Case Illustrations== | ==Case Illustrations== | ||
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[[Image:Fgij-xl10-histyr-clash.png]] | [[Image:Fgij-xl10-histyr-clash.png]] | ||
</td></tr></table> | </td></tr></table> | ||
[[4rea]] also has a clash suggested as a possible isopeptide bond by FirstGlance. Both that, and the His-Tyr clashes are removed by [[PDB REDO]]. | |||
===Thioesters=== | ===Thioesters=== | ||
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</span> | </span> | ||
</td></tr></table> | </td></tr></table> | ||
[[2xi9]] has 2 copies of a bacterial pilus adhesin in its [[asymmetric unit]]. FirstGlance reports a putative thioester crosslink in each copy of the protein. The relevant residues are modeled as a thiester bond with correct geometry. The C-S distances in the model are 1.6 Å, in good agreement with the typical bond length of 1.7 Å<ref name="bondlengths" />. The electron density map shows clear density for the thioester-bonded atoms. The difference map (not shown) raises no alarms. The abstract of the publication highlights this thioester bond. | [[2xi9]] has 2 copies of a bacterial pilus adhesin in its [[asymmetric unit]]. FirstGlance reports a putative [[Thioester protein crosslinks|thioester crosslink]] in each copy of the protein. The relevant residues are modeled as a thiester bond with correct geometry. The C-S distances in the model are 1.6 Å, in good agreement with the typical bond length of 1.7 Å<ref name="bondlengths" />. The electron density map shows clear density for the thioester-bonded atoms. The difference map (not shown) raises no alarms. The abstract of the publication highlights this thioester bond. | ||
===="Thioester" Clash==== | ===="Thioester" Clash==== | ||
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[[5d12]] has two copies of a kinase domain in its [[asymmetric unit]]. FirstGlance reports a possible thioester crosslink only in chain B. The C-S distance is 1.76 Å, quite reasonable. However, the residues are not modeled as a thiester bond, and Jmol draws many spurious "bonds". The sidechain of Asp348 passes through a gap in the all-features electron density (<span style="color:#00a000;font-weight:bold;">green isomesh</span>, above left). The difference electron density map (above right) reveals insufficient density to account for the position of the Asp sidechain (<span style="color:#b00000;font-weight:bold;">dark red isomesh</span>), while suggesting an alternative position for those sidechain atoms (<span style="color:#0000ff;font-weight:bold;">blue isomesh</span>). In conclusion, this is a clash, not a thioester crosslink bond. | [[5d12]] has two copies of a kinase domain in its [[asymmetric unit]]. FirstGlance reports a possible thioester crosslink only in chain B. The C-S distance is 1.76 Å, quite reasonable. However, the residues are not modeled as a thiester bond, and Jmol draws many spurious "bonds". The sidechain of Asp348 passes through a gap in the all-features electron density (<span style="color:#00a000;font-weight:bold;">green isomesh</span>, above left). The difference electron density map (above right) reveals insufficient density to account for the position of the Asp sidechain (<span style="color:#b00000;font-weight:bold;">dark red isomesh</span>), while suggesting an alternative position for those sidechain atoms (<span style="color:#0000ff;font-weight:bold;">blue isomesh</span>). In conclusion, this is a clash, not a thioester crosslink bond. | ||
After [[PDB REDO]] re-refines and rebuilds 5d12, this clash is absent. | |||
===Lys-Cys NOS=== | ===Lys-Cys NOS=== | ||
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[[7ns5]] is a homo-tetramer. FirstGlance suggests a possible Lys-Cys NOS crosslink in chain B. The fact that FirstGlance suggests no such crosslinks in the other 3 chains is a hint that the proximity of the sidechain nitrogen of Lys167 to the sulfur in Cys165 in chain B is a clash. The atomic model shows an improbable N-S bond between the sidechains. Further, electron density (above left) is missing for the last 3 atoms of the lysine sidechain, consistent with it being misplaced (<span style="color:#00a000;font-weight:bold;">green isomesh</span>). The difference electron density map (above right) reveals that there is no electron density consistent with the lysine sidechain nitrogen being located where it was placed in the model (<span style="color:#b00000;font-weight:bold;">dark red isomesh</span>). In conclusion, this is a clash in chain B, not an NOS crosslink. | [[7ns5]] is a homo-tetramer. FirstGlance suggests a possible Lys-Cys NOS crosslink in chain B. The fact that FirstGlance suggests no such crosslinks in the other 3 chains is a hint that the proximity of the sidechain nitrogen of Lys167 to the sulfur in Cys165 in chain B is a clash. The atomic model shows an improbable N-S bond between the sidechains. Further, electron density (above left) is missing for the last 3 atoms of the lysine sidechain, consistent with it being misplaced (<span style="color:#00a000;font-weight:bold;">green isomesh</span>). The difference electron density map (above right) reveals that there is no electron density consistent with the lysine sidechain nitrogen being located where it was placed in the model (<span style="color:#b00000;font-weight:bold;">dark red isomesh</span>). In conclusion, this is a clash in chain B, not an NOS crosslink. | ||
After [[PDB REDO]] re-refines and rebuilds 7ns5, this clash is absent. | |||
==See Also== | |||
*[[FirstGlance/Index]]: FirstGlance resources within and external to Proteopedia. | |||
==Notes and References== | ==Notes and References== | ||
<references /> | <references /> | ||